Isolation, expression and biochemical characterization of recombinant hyoscyamine-6β-hydroxylase from Brugmansia sanguinea - tuning the scopolamine production

Hyoscyamine-6-hydroxylase (H6H, EC 1.14.11.11) is a plant enzyme that catalyses the last two steps in the biosynthesis of the anticholinergic drug scopolamine, i.e. the hydroxylation of hyoscyamine to 6-hydroxyhyoscyamine (anisodamine) and subsequent oxidative ring-closure to the 6,7--epoxide. A H6H gene homologue was isolated from the plant Brugmansia sanguinea (BsH6H) and recombinantly cloned into Escherichia coli, expressed and purified using an effective SUMO-fusion procedure. Enzymatic activity is approximately 40-fold higher for the first reaction step and the substrate affinity is comparable to other characterized H6H homologues (K-m approximate to 60 M). Truncation of an H6H enzyme flexible N-terminal region yields an active and stable yet more compact enzyme version.